Fowler, Jane

Gülay, Arda

Palomo, Alejandro

Rasmussen, Simon

Schramm, Andreas

Sicheritz-Pontén, Thomas

Smets, Barth F.

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Dec 18, 2017Investigating comammox Nitrospira in rapid sand filters via metagenomics and single-cell genomicsPalomo, Alejandro; Fowler, Jane; Gülay, Arda; Rasmussen, Simon; Schramm, Andreas; Sicheritz-Pontén, Thomas; Smets, Barth F.Published in:Microbial ecology and water engineering &amp; biofilms specialist groups (MEWE2016)Publication date:2016Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Palomo, A., Fowler, J., Gülay, A., Rasmussen, S., Schramm, A., Sicheritz-Pontén, T., & Smets, B. F. (2016).Investigating comammox Nitrospira in rapid sand filters via metagenomics and single-cell genomics. In Microbialecology and water engineering & biofilms specialist groups (MEWE2016) (pp. 20-21). Copenhagen, Denmark :IWA.0/"!4!/!!26!.!4\6)M_6)DZXY^D02!/)(!/D!/.4,Session 2: Systems biology approaches for assessing microbial communities in water engineering I  IWA Specialist Conference Microbial Ecology and Water Engineering 2016:  A joint conference of the MEWE and Biofilm specialist groups 20 Investigating comammox Nitrospira in rapid sand filters via metagenomics and single-cell genomics   Alejandro Palomo1,2, Jane Fowler1, Arda Gülay1, Simon Rasmussen2, Andreas Schramm3, Thomas Sicheritz-Ponten2, Barth F. Smets1  1Department of Environmental Engineering, Technical University of Denmark, Kongens Lyngby, Denmark. 2Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark 3Center for Geomicrobiology, Department of Bioscience, Aarhus University, Aarhus, Denmark * Email: alpal@env.dtu.dk  Keywords: nitrification, comammox Nitrospira, metagenomics, single cell genomics Introduction Throughout the last century, nitrification was assumed to be a two-step process executed by two different functional groups, ammonia oxidizing prokaryotes (AOP) and nitrite oxidizing bacteria (NOB). Recently, several articles have shown the capability of a single microorganism (belonging to the Nitrospira genus) to carry out the complete oxidation of ammonia to nitrate (comammox) (Daims et al., 2015; van Kessel et al., 2015; Pinto et al., 2015, Palomo et al., 2016). Despite the detection and enrichment of commamox Nitrospira from different nitrifying environments, the ecological relevance of comammox remains unknown. Nitrification is often the dominant microbial process in rapid gravity sand filters (RSF), which are engineered microbial systems used for the production of drinking water.  High abundance of Nitrospira and unusual Nitrospira/AOP ratios have been observed in these systems (Albers et al., 2015; LaPara et al., 2015, Gülay et al., 2016). In this study, we applied a combination of metagenomics and single-cell genomics to microbial communities from various locations within an aerated groundwater-fed RSF, where Nitrospira are present at high relative abundance (Gülay et al., 2016).  Material and Methods Genomic DNA was extracted from six different locations within a groundwater-fed rapid sand filter. DNA was sequenced using Illumina HiSeq 2000 giving 100 bp paired-end reads. High quality reads were assembled using IDBA-UD. Contigs larger than 1,000 nucleotides were clustered into putative taxonomic groups based on pentanucleotide signatures using VizBin. Nitrospira single cell sorting was achieved using an in-house developed cell extraction strategy that enabled the disruption of Nitrospira cell clusters attached to the mineral coating of the sand. Extracted cells were identified via fluorescence in situ hybridization (FISH) with Nitrospira-specific 16S rRNA probes and were sorted via fluorescence-activated cell sorting (FACS).  Genomic DNA of sorted cells was amplified with multiple displacement amplification (MDA), and screened using Nitrospira-specific 16S rRNA gene primers. Selected Nitrospira genomes were subject to whole-genome sequencing. Results and Conclusions From the metagenomic data set, fourteen draft genomes were reconstructed and functionally characterized. The organisms represented by draft genomes had the capability to oxidize ammonium, nitrite, hydrogen sulfide, methane, potentially iron and manganese as well as organic compounds. Interestingly, a highly abundant microdiverse composite genome of the Nitrospira genus was recovered harboring the genetic potential for complete ammonia oxidation. To evaluate the potential relevance of comammox Nitrospira in the removal of ammonium within the studied RSF, we investigated the phylogenetic diversity and abundance of amoA as a marker gene of ammonium oxidation. The RSF metagenome amoA sequences separated into 4 different clusters. Themajority of the amoA genes were associated with comammox Nitrospira (81% and 52% in top and bulk). Seven and 1% (top and bulk filter,respectively) of amoA genes were related to a cluster of canonical beta-proteobacterial AOB. Session 2: Systems biology approaches for assessing microbial communities in water engineering I  IWA Specialist Conference Microbial Ecology and Water Engineering 2016:  A joint conference of the MEWE and Biofilm specialist groups 21 AOA-associated amoA were rarely detected, making up less than 1% in top and bulk samples. amoA associated with heterotrophic ammoniaoxidizers made up 12% and 46% of all amoA (top and bulk filter,respectively). (Figure 1).   Cell extraction from sand samples followed by FACS and single-cell sequencing enabled assembly of Nitrospira genomes from the RSF (Figure 2). Single cell genomes confirmed the presence of a complete ammonia oxidation pathway in Nitrospira and revealed clear taxonomic differences between comammox Nitrospira in the investigated RSF and other  recently described comammox Nitrospira genomes. The high abundance of comammox Nitrospira spp. and the dominance of Nitrospira-associated amoA sequences in the metagenome suggest an essential role in ammonia oxidation of this novel comammox Nitrospira in the investigated RSF.   ReferencesAlbers CN, Ellegaard-Jensen L, Harder CB, Rosendahl S, Knudsen BE, Ekelund F, et al. (2015). Groundwater Chemistry Determines the Prokaryotic Community Structur of Waterworks Sand Filters. Environ Sci Technol 49: 839–846. Daims H, Lebedeva E V, Pjevac P, Han P, Herbold C, Albertsen M, et al. (2015). Complete nitrification by Nitrospira bacteria. Nature 528: 504–509. Gülay A, Musovic S, Alberchtsen H-J, Al-Soud WA, Sørensen S, Smets BF. (2016). Ecological patterns, diversity and core taxa of microbial communities in groundwater- fed rapid gravity filters. ISME J doi:10.1038/ismej.2016.16 LaPara TM, Hope Wilkinson K, Strait JM, Hozalski RM, Sadowksy MJ, Hamilton MJ. (2015). The bacterial communities of full-scale biologically active, granular activated carbon filters are stable and diverse and potentially contain novel ammonia-oxidizing microorganisms. Appl Environ Microbiol 81: 6864–6872. Palomo A, Fowler SJ, Gülay A, Rasmussen S, Sicheritz-Ponten T, Smets BF. (2016) Metagenonic analysis of rapid gravity sand filter microbial communities suggests novel physiology of Nitrospira spp.. ISME J, Accepted. Pinto AJ, Marcus DN, Ijaz Z, Bautista-de los Santos QM, Dick GJ, Raskin L. (2015). Metagenomic Evidence for the Presence of Comammox Nitrospira-Like Bacteria in a Drinking Water System. mSphere 1: e00054–15. van Kessel MAHJ, Speth DR, Albertsen M, Nielsen PH, Op den Camp HJM, Kartal B, et al. (2015). Complete nitrification by a single microorganism. 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Investigating comammox Nitrospira in rapid sand filters via metagenomics and single-cell genomics

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Investigating comammox Nitrospira in rapid sand filters via metagenomics and single-cell genomics

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